3′-amino-3′-deoxy adenosine was enzymatically converted into 3′-amino-3′-deoxy ATP. This ATP analogue was incorporated into the 3′-terminal adenosine position of Escherichia coli tRNA. The modified tRNA was aminoacylated with phenylalanine by use of E. coli phenylalanyl tRNA synthetase. The phenylalanine was attached to the 3′-amino group of the tRNA, as shown by its high resistance to base-catalyzed hydrolysis in contrast with the normal lability of phenylalanyl-tRNA. Aminoacyl tRNA synthetases charge the 3′-amino-3′-deoxy tRNA with kinetics that are similar to those of the charging reaction in which normal tRNA is the substrate. When phenylalanyl-3′-amino-3′-deoxy tRNA is used in a protein-synthesizing system directed by poly(U) in vitro, this molecule is capable of receiving an acetyl-phenylalanine from the donor site of the ribosome. However, the ribosome is unable to cleave the amide bond connecting phenylalanine to the tRNA molecule; hence, the phenylalanyl-3′-amino-3′-deoxy tRNA has acceptor but not donor activity in protein synthesis. Failure of the ribosome to cleave the amide bond may be due to its greater stability relative to the normal ester bond. However, it may also be due to the fact that the isomerization of the peptidyl chain between the 2′ and 3′ positions of adenosine is prevented due to the amide bond at the 3′ position, and cleavage may normally occur with the peptidyl chain on the 2′ position of adenosine.